Positively regulated, OmpR-controlled promoter from OmpC gene (R0082) directing transcription of P22 c2 gene. The P22 c2 protein represses expression of mRFP (M30002). Constructed with standard assembly method except R0082 was isolated using VF and VR-directed PCR. Consequently, there may be silent mutations that were inadvertently introduced. A second isolate from this construction was saved as M30008.
Cascaded Tet (medium) and Lac (weak) QPIs with double terminator as input
This component tests the BBa_R0080 AraC-induced promoter region.
The second screening plasmid. Same as the first, except with stronger RBSs. Ends should be capped with I13450.
Cascaded Lac (elowitz) and Tet (medium) QPIs with constitutive promoter as input
Parts I13007-I130014 are RBS test constructs for RBS characterization. Each consists of Lac or Tet promoter, one of the four RBS's B0030-B0033, a YFP coding protein, and B0015 terminator.
The fourth screening plasmid. Same as the second, except without an insertion site. Sole purpose is to check whether the insertion site alone affects the performance. Ends should still be capped with I13450.
Cascaded Tet (medium) and Lac (weak) QPIs with constitutive promoter as input
Microbes including Escherichia coli are known to respond to various DNA-injuring stress (ionizing radiation, ultraviolet radiation, peroxides etc...), altering their gene expressions. This response, known as "SOS response", are induced by regulatory protein called RecA, which is activated when binds to single-strand DNA. DNA-RecA complex promotes the self-degradation of LexA, a common repressor for SOS genes. SulA is responsible for stress-induced halt of cell division. The promoter of sulA, or sulAp, is acutely induced by various stress factors, including ultraviolet irradiation. We provide a sulAp evaluation device to make it easy to measure the varying expression of sulAp in various "stressful" conditions. Employing a dual luciferase reporter construct, quantitative measurement and comparison of Relative Promoter Unit (RPU) is achieved.
sulAp + dual luciferase reporter construct
sulAp (BBa_K518010) was cloned from Escherichia coli K12 strain's genome.
Cascaded Lac (medium) and Tet (medium) QPIs with double terminator as input
Cascaded Tet (weak) and Lac (medium) QPIs with constitutive promoter as input
This part tests the effecacy of the I12224 protein generator.
Positively regulated, OmpR-controlled promoter from OmpC gene (R0082) directing transcription of lambda cI gene. The cI protein represses expression of mRFP (M30003). Constructed with standard assembly method except R0082 was isolated using VF and VR-directed PCR. Consequently, there may be silent mutations that were inadvertently introduced.
R0040.B0032.E0040.B0015, GFP expression device
phoApis a phosphate-responsive promoter. In high concentration of phosphate, it will be repressed while in low phosphate concentration, it will be activated.
phoAp is native in E. coli
PkdpF[-15, T>A] - GFP generator
PhoA (E.coli JM83 alkaline phosphatase gene)
phoA can substituted for the periplasmic region of ToxR transmembrane dimerizing protein. igemmit2005 will be using this as a positive control in tests related to ToxR pathway.
Cascaded Lac (medium) and Tet (medium) QPIs with constitutive promoter as input
Positively regulated, OmpR-controlled promoter from OmpC gene (R0082) directing transcription of P22 c2 gene. The P22 c2 protein represses expression of mRFP (M30001). Constructed with standard assembly method except R0082 was isolated using VF and VR-directed PCR. Consequently, there may be silent mutations that were inadvertently introduced. A second isolate from this construction was saved as M30005.
Reporter Device Powered by the Virtual Machine (VM1.0)
Simple GFP generator but controlled by an orthogonal promoter (T7 consensus) and an orthogonal RBS (Brink et al.) Will only produce a high output in a T7 gp1.0 and sRBS strain.
Cascaded Lac (medium) and Tet (elowitz) QPIs with double terminator as input
This part contains: - a mutated ''virG'' response regulator from the virA/G receptor system from ''Agrobacterium tumefaciens'' which works in ''Escherichia coli'' without an ''rpoA'' gene from ''A. tumefaciens'' - a kanamycin resistance under the control of a ''vir'' promotor This device is used to screen ''virA'' receptor mutants. The ''virA'' receptor is mutated in the BBa_K389010 BioBrick. The better the receptor detects a substance, the more ''virG'' is phosphorylated and the stronger the kanamycin resistance from this BioBrick is expressed. The BioBricks BBa_K389010 and BBa_K389011 have to be used together in one cell.
medium strong constitutive promotor, terminator to keep basal expression of kanamycin resistance low
synthetic ''virG'' gene, ''vir'' promotor from ''A. tumefaciens'' C58, partsregistry
Positively regulated, OmpR-controlled promoter from OmpC gene (R0082) directing transcription of mRFP (I13507). Constructed with standard assembly method except R0082 was isolated using VF and VR-directed PCR. Consequently, there may be silent mutations that were inadvertently introduced. An alternative clone is M30011. Cells bearing this construct are pale pink.
σx Generator + PcomFA-E0240
Ng Siu Wang Edward, Jason Jang, Joo Min Jung
SigmaX generator, SigmaX-inducible Phelicase encoding GFP
Both comX gene and Phelicase are found in genomic sequence of Streptococcus pneumoniae NCTC7465 strain, while the RBS+GFP+DTT, downstream of PCelA, is BBa_E0240. The promoter encoding comX is BBa_K880005 and the double terminator for it is BBa_B0015
Low output control (tet QPI, Elowitz RBS)
GFP induced by micro-aerobic promoter vgb
This part consists of promoter vgb, RBS and GFP coding sequence. Promoter vgb is induced under micro-aerobic condition. Activated by the promoter, GFP coding gene expresses and the florescence intensity could represent expression rate of GFP to some degrees, by which we can confirm the character of this promoter.
Which restriction sites should be choosed to promise the high efficiency of assembling. Whether the linked product have capability to measure the expression of promoter vgb.
All subparts are obtained in 2014 Distribution. All of them are designed and assembled by students.
R0040.B0030.E0040.B0015, GFP expression device
Sensing Device A (cI) TESTER (Pr+GFP+Prm+RFP)
A debug version of the event sensing and processing system for the iGEM2005 ETH "Counter" project: its purpose is to regulate cI production dependent on an external event. It works somehow like an inverter: STATE 1: when IPTG is absent, the constitutively produced lacI protein will block the lacI+pL promoter so that no cI is produced. Thus downstream the Pr promoter will be constitutively active, producing GFP (green fluorescence), while Prm+ will have low or no activity (no/low RFP - red fluorescence). STATE 2: when IPTG is present, it will remove the lacI protein from the lacI+pL promoter and cI is produced. Thus downstream the Pr promoter will be repressed (no GFP, i.e. no green fluorescence) by cI, while Prm+ will be activated, producing RFP (red fluorescence).
For more details see Sensing Device and Event Processing Device documentation.
PkdpF[-15, T>G] - GFP generator
Tester to investigate zinc finger proteins (ZFP) as a negative regulator of expression. Lac promoter controls expression of a ZFP and a reporter (i.e. RFP) to confirm expression. Lambda promoter constitutively expresses GFP unless ZFP binds to the binding site after the lambda promoter.
Promotor RBS Zinc_Finger RBS RFP Terminator Promotor ZF-Binding_site1 ZF-Binding_site2 RBS GFP Terminator
Cascaded Tet (elowitz) and Lac (medium) QPIs with constitutive promoter as input
Positively regulated, OmpR-controlled promoter from OmpC gene (R0082) directing transcription of mRFP (I13507). Constructed with standard assembly method except R0082 was isolated using VF and VR-directed PCR. Consequently, there may be silent mutations that were inadvertently introduced. An alternative clone is M30012. Cells bearing this construct are pale pink.
Positively regulated, OmpR-controlled promoter from OmpC gene (R0082) directing transcription of P22 c2 gene. The P22 c2 protein represses expression of mRFP (M30001). Constructed with standard assembly method except R0082 was isolated using VF and VR-directed PCR. Consequently, there may be silent mutations that were inadvertently introduced. A second isolate from this construction was saved as M30006.
This component tests the I12222 TetR protein generator. It uses the R0080 promoter region for the test.
Cascaded Lac (elowitz) and Tet (medium) QPIs with double terminator as input
PcelA-E0240 (Measurement Kit for PcelA)
Jordy Evan, Nadia Benedicta, Angeline Widjaja
SigmaX-dependent promoter initiating the transcription of GFP
The promoter is found genomic sequence of Streptococcus pneumoniae NCTC7465 strain, while the RBS+GFP+DTT is BBa_E0240
PcomFA-E0240 (Measurement Kit for PcomFA)
Positively regulated, OmpR-controlled promoter from OmpC gene (R0082) directing transcription of P22 c2 gene. The P22 c2 protein represses expression of mRFP (M30002). Constructed with standard assembly method except R0082 was isolated using VF and VR-directed PCR. Consequently, there may be silent mutations that were inadvertently introduced. A second isolate from this construction was saved as M30007.
R0040.B0030.E0044.B0015, GFP-AAV expression device
VirA/G signaling system + sensitivity tuner + luciferase
This part combines a complete VirA/G signaling system with a sensitivity tuner before a luciferase gene under the control of a ''vir'' promoter. The sensitivity tuner enhances a signal from a promoter so it enhances the readout luciferase.
sensitivity tuner to enhance the luciferase signal
* luciferase from pGL4.10luc2 (Promega) * sensitivity tuner part, terminators and constitutive promoter from the partsregistry * ''virG'' was synthesized by Mr. Gene * ''virA'' and ''vir'' promoter from ''Agrobacterium tumefaciens'' C58, TI-plasmid
Cascaded Tet (elowitz) and Lac (medium) QPIs with double terminator as input
GFP - LacZ protein generator is generated with the aim of inducing a reporting reaction aftermath of the activation of EpCAM detection system. (BBa_K772100) For more information: BBa_K772006
To achieve a constitutive production, pTetR promoter is added in the upstream region of the original part.
Cleavage site, LacZ and GFP sequences are gathered from Registry.
Low output control (lac QPI, Elowitz RBS)
Cascaded Lac (medium) and Tet (weak) QPIs
GFP - LacZ protein complex with flag tags
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, a reporter device (LacZ enzyme catalyses the reaction of the cleavage of X-Gal which results with blue color change) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system. This part contains flag tags extra from BBa_K772006 in the GFP region; because it is planned to be used in the Co-IP/SDS-PAGE assay to be isolated.
Note that there is an RBS sequence just before the protein sequence in order to eliminate the need of addition of it.
R0040.B0032.E0044.B0015, GFP-AAV expression device
Low output control (c2 QPI, Elowitz RBS)
Cascaded Lac (medium) and Tet (elowitz) QPIs with constitutive promoter as input
ToxR'::PhoA (with promoter and RBS and terminator)
This part is a positive control for ToxR' dimerization.
Cascaded Tet (medium) and Lac (weak) QPIs
Firefly-renilla dual luciferase assay kit
Luciferase assay is one of the most popular reporter assay system for quantitatively measuring the function of promoters and other cis-elements. The wide-ranging and quantitative detection is the prominent feature of this assay; light emmision from 10~(-18) ― 10~(-11) mol protein can be quantitatively measured using luminometer (see our results). A promoter or other cis-element to be analysed can be ligated above this part. Its effect will be measured as an altered firefly luciferase expression. Meanwhile, renilla luciferase expression can be used to as an internal control.
Low output control (c1 QPI, Elowitz RBS)
The first screening plasmid. Ends will be capped with I13450. Cutting I13452 generates an internal BioBrick site.
YFP tagged with an AAV ssrA tag with transcription initiated from pTet
Sensing Device B (cI+LVA) TESTER (Pr+GFP+Prm+RFP)
A debug version of the event sensing and processing system for the iGEM2005 ETH "Counter" project: its purpose is to regulate cI+LVA (degradation tag) production dependent on an external event. It works somehow like an inverter: STATE 1: when IPTG is absent, the constitutively produced lacI protein will block the lacI+pL promoter so that no cI is produced. Thus downstream the Pr promoter will be constitutively active, producing GFP (green fluorescence), while Prm+ will have low or no activity (no/low RFP - red fluorescence). STATE 2: when IPTG is present, it will remove the lacI protein from the lacI+pL promoter and cI is produced. Thus downstream the Pr promoter will be repressed (no GFP, i.e. no green fluorescence) by cI, while Prm+ will be activated, producing RFP (red fluorescence).
YFP with an AAV ssrA tag with transcription initiated by LuxpL
GFP - LacZ anchor-inducer protein complex
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, a reporter/inducer device or protein (enzyme etc.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the inducer/reporter. In conclusion, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system.
Cascaded Tet (medium) and Lac (medium) QPIs
PkdpF[-15, T>C] - GFP generator
This part combines a complete VirA/G signaling system with a sensitivity tuner before a luciferase gene under the control of a vir promoter. The sensitivity tuner enhances a signal from a promoter so it enhances the readout luciferase.
* luciferase from pGL4.10luc2 (Promega) * sensitivity tuner part, terminators and constitutive promoter from the partsregistry * ''virG'' was synthesized by Mr. Gene * ''virA'' and ''vir'' promoter from Agrobacterium tumefaciens C58, TI-plasmid
Cascaded Lac (elowitz) and Tet (medium) QPIs
With this system it is possible to measure the activity of a virA receptor and the vir promoter, respectively. The reporter is the mRFP from the partsregistry.
medium strong constitutive promotor, terminator to keep basal expression of mRFP low
partsregistry, synthetic gene by Mr. Gene, TI-plasmid from ''Agrobacterium tumefaciens'' C58
Cascaded Tet (weak) and Lac (medium) QPIs
Cascaded Lac (medium) and Tet (medium) QPIs
Cascaded Tet (medium) and Lac (medium) QPIs with double terminator as input
ToxR'::MalE(with promoter and RBS and terminator)
This part is a negative control for ToxR' dimerization. MalE only exists as a monomer, and when fused to ToxR', will prevent it from dimerizing.
This component tests the I12222 TetR protein generator. It uses the I0500 promoter region for the test.
The third screening plasmid. Same as the second except with the flurophores switched. The ends should be capped with I13450.
Cascaded Tet (weak) and Lac (medium) QPIs with double terminator as input
Cascaded Lac (medium) and Tet (elowitz) QPIs
Cascaded Tet (medium) and Lac (medium) QPIs with constitutive promoter as input
R0011.E0840 (S_N; strong rbs; high PoPS)
Lambda-lacI input system TESTER (IPTG controlled lacI+pL efficiency)
Constitutive (tetR) lacI-production (lacI+LVA) to block promoter (lacI+pL) in ground state of a 2-state system. lacI+pL can be activated, e.g. over IPTG, thus leading to the production of GFP (green fluorescence). This device allows to estimate repressed (lacI) activity/full activity under ITPG absence/presence.
Cascaded Lac (elowitz) and Tet (weak) QPIs
With this system it is possible to measure the activity of a ''virA'' receptor and the ''vir'' promoter, respectively. The reporter is a firefly luciferase from Promega's pGL4.10luc2 plasmid.
medium strong constitutive promotor, terminator to keep basal expression of luciferase low
partsregistry, synthetic gene by Mr. Gene, pGL4.10luc2 by Promega, TI-plasmid from ''Agrobacterium tumefaciens'' C58
Cascaded Lac (weak) and Tet (medium) QPIs
σx Generator + PcelA-E0240
SigmaX generator, SigmaX inducible PCelA encoding GFP
Both comX gene and PCelA are found in genomic sequence of Streptococcus pneumoniae NCTC7465 strain, while the RBS+GFP+DTT which is downstream of PCelA is BBa_E0240. The promoter encoding comX is BBa_K880005 and the double terminator for it is BBa_B0015
Cascaded Tet (elowitz) and Lac (medium) QPIs
This component tests the I12222 TetR protein generator.